The isolation of biomolecules is a critical part of any sample processing system. With the development of automated molecular analysis systems the biggest restriction is in the preparation of the sample and the purification of the target sample.
For all biochemistry processes the isolation and purification of the sample target is critical to its success. The limitations in biochemistry analysis process—pyro-sequencing, nucleic acid ligation, polymerase chain reaction, digital PCR, qPCR, nucleic acid sequencing, protein detection/protein enrichment, genetic bead coating, rare cell detection and cell enrichment—and not limited to these, are due to the starting concentrations of the target and the level of biochemical inhibitors present within the reaction sample used in the analysis.
For most biochemistry analyses a series of pre-analysis steps are performed on the sample to isolate the target from the initial sample and remove biochemistry inhibitors. These steps are typically labor intensive and ultimately reduce the starting concentrations of the target.
The current preferred method of sample purification makes use of spin columns. However spin columns require a number of centrifugation steps and hence cannot be integrated with an automated DNA library preparation platform. Similarly, a purification technique for nucleic acid fragment purification from agarose gels also requires centrifugation steps to achieve the nucleic acid isolation.
One technique used for sample purification is paramagnetic bead-based purification. This method offers an approach that can provide improved DNA recovery rates and tuneable buffer conditions that can be used to selectively bind specific DNA fragment sizes.
The paramagnetic bead based purification is a static well batch process. The current method involves the pipetting of the bead-mixture—paramagnetic beads and a buffer—into a well of a microtitre plate along with the initial sample. The solution is pipetted, mixed, and incubated at room temperature to allow the DNA to bind to the beads. The microtitre plate is then placed onto a magnetic plate. The beads holding the bound DNA move to the edge of the plate and are held by the magnet. Next the supernatant (containing waste) is removed using a pipette and discarded. Following this a number of wash steps are then performed to remove residual waste present on/at the bead pellet. Ethanol is pipetted to the plate containing the bead pellet, incubated and then removed using a pipette. This wash step is repeated twice. An elution buffer is then added. The plate is removed from the magnetic plate and the elution buffer is mixed via pipette mixing. The microtitre plate is placed back onto the magnetic plate. The eluent containing the purified DNA is then withdrawn using a pipette.
The paramagnetic bead based protocol is a labour intensive process and is not easily automated due to the large number of pipetting steps required. The high numbers of pipetting steps also result in large initial and final sample volumes, resulting in high reagent costs per data point.
One application and not limited to this application is for improved sample purification for next generation sequencing platforms. Many next generation sequencing platforms require DNA libraries made up of DNA fragments within a specific range of base pair lengths. In addition, these DNA fragments need to be tagged with specific nucleotide sequences (adapters) to allow the sequences to be amplified using PCR and to allow the library fragments to anneal to the sequencer flow cell. Sequence specific indices can also be added to the DNA fragments to identify individual samples when multiplexing sample within a single flow cell. The tagmentation of DNA (DNA is fragmented and tagged with adapters) and the addition of common adapters and indices are achieved in two separate biological reactions. Following these reactions, the DNA library is cleaned to remove excess nucleotides, enzymes, primers, salts and other contaminants. Consequently, the workflow required to tagment DNA, purify tagmented DNA, add common adapters and indices and purify the final library product is complex and labour intensive.
The systems and methods outlined herein can help achieve sample handling that is contamination-free, low-volume, high-throughput, low-cost, and/or high in sample concentration.